Agaricus subrufescens mushroom plant named ‘H1X1’

ABSTRACT

A new and unique variety of the mushroom  Agaricus subrufescens  Peck was produced by crossbreeding a single spore isolate from the fungal strain ‘I-101’ and a single-spore isolate from the fungal strain ‘SBRFG’. The resultant hybrid, named ‘H1X1’, exhibits early cropping, large size, high productivity, and an attractive appearance that includes a smooth, round cap.

Latin name of genus and species: Agaricus subrufescens Peck.

Varietal denomination: ‘H1X1’.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinct variety of mushroomplant of Agaricus subrufescens Peck. The present plant is an ediblemushroom.

Agaricus subrufescens was first described in 1893 by C. H. Peck, the NewYork State Botanist, from mushrooms being cultivated in a greenhouse inDosoris, N.Y., and some additional specimens found growing in the woodsnearby. The mushroom, nicknamed the ‘almond mushroom’ due to itsfragrance and taste, was widely cultivated, sold, and eaten in theAtlantic states of the United States of America from at leastMassachusetts to Washington, D.C. in the latter years of the 19^(th)century. Spawn (i.e., inoculum culture for farming) of Agaricussubrufescens was even offered for sale in catalogs of the day.Subsequently, commercial production of Agaricus subrufescens declined asmarket trends changed, and by the 1920-1930 period, virtually the onlymushroom species being cultivated in the United States was the related‘button mushroom’ species, Agaricus bisporus (Lange) Imbach. Agaricussubrufescens mushrooms have occasionally been found growing wild outsideof northeastern North America in place such as, for example, California,Israel, and Hawaii. The recently described mushroom Agaricus rufotegulisNauta from the Europe is, based on ITS1+2 and other DNA sequences,actually Agaricus subrufescens, and the same is true of the mushroomAgaricus brasiliensis Wasser et al. from South America.

The history of the mushroom Agaricus subrufescens, as well as adiscussion of its properties as an easily cultivated mushroom has beenreviewed and present by Kerrigan, one of the inventors of record, inseveral different articles circa 1983-1984. A culture of Agaricussubrufescens was isolated by Kerrigan from soil in California in 1982and was subsequently sold commercially to hobbyist mushroom growers.Reproductive mode(s) and genetic behavior of this strain wereinvestigated by Kerrigan and Ross (1987), and Kerrigan (2005).

Agaricus subrufescens mushrooms have more recently begun to becultivated again on a wide scale, particularly in Brazil and Japan, as a‘medicinal mushroom’ that is marketed primarily in Japan. However, inthis context, the mushroom is typically referred to (incorrectly) as (1)Agaricus blazei Murr., (2) Agaricus blazei Marr. sensu Heineman, (3)Agaricus sylvaticus, or the recently coined (4) Agaricus brasiliensis.However, the data presented below and in Kerrigan, “Agaricussubrufescens, a cultivated edible and medicinal mushroom, and itssynonyms,” Mycologia, 97(1), May 18, 2005, pp. 12-24, the disclosure ofwhich is incorporated herein by reference, indicate that the medicinalmushroom from Brazil and Japan is biologically and phylogenetically thesame species as Agaricus subrufescens from North America. As Peck'sspecies name Agaricus subrufescens is older than either Argaricusbrasilensis or Agaricus rufotegulis, it has a nomenclatural priorityunder the International Code of Botanical Nomenclature and is thereforethe correct name of the species. Therefore, the name Agaricussubrufescens is used herein inclusively. The species name applies notonly to phylogenetically congruent isolates and specimens from all partsof the world but also to hybrids between them.

Cultivated material of Agaricus subrufescens was obtained fromCalifornia, Brazil, Japan, and China. Hybridization (crossing)experiments were performed on selected pairs of stocks. Whensingle-spore isolates (SSIs) from two different stocks are successfullycrossed, the resulting hybrid incorporates genetic material from bothprogenitors, and the traits inherited from the parents may exist in anovel combination, or be intermediate in their nature. The successfulproduction of hybrids strongly supports treatment of the parentalisolates as members of a single species, regardless of their origin.

Furthermore, rDNA ITS1+2 sequences from many strains of Agaricussubrufescens were obtained from North America, South America, Europe,and Hawaii. It has been observed that Agaricus subrufescens has aconsiderable degree of cultural and morphological variability, but thisvariability is not vastly greater than in other species of Agaricusincluding Agaricus bisporus. It has also been found that the DNAsequences of diverse Agaricus subrufescens cultures are either identicalor extremely similar, as is typically the case within other species ofAgaricus, and share characteristics polymorphisms not known from otherspecies. Thus, molecular markers have been developed that identify thespecies, identify strains within the species, and document thetransmission of hereditary material from parental strains intooffspring.

SUMMARY OF THE INVENTION

The present invention is a new and distinct variety of Agaricussubrufescens mushroom characterized by its rapid growth, highproductivity, robust size and stature, and smooth round cap. That is,the present mushroom plant may be characterized by early and abundantproduction of mushrooms which are large and fleshy, with smooth, roundedcaps. The new mushroom also has a genotype that combines markers fromeach of its progenitors. This novel and distinct variety of mushroom isidentified as a Agaricus subrufescens hybrid mushroom named ‘H1X1’. Thisnew hybrid mushroom variety was produced in the breeding program ofSylvan Research, 198 Nolte Dr., Kittanning, Pa. 16201, by crossbreedinga single-spore isolate from the fungal strain ‘I-101’ and a single-sporeisolate from the fungal strain ‘SBRFG’.

The Agaricus subrufescens mushroom has been asexually reproduced bymeans of mycelial propagation in Kittanning, Pa. The resulting transferculture (or ‘subculture’) is a common method for maintaining a mushroomstrain on suitable media. Media such as PDA or MEA can be used as themushroom culture media and the strain can be subcultured as frequentlyas every two or three weeks. To vegetatively propagate the mushroomculture aseptically, under laboratory conditions, a small portion of apure (=axenic) culture on a suitable medium, such as potato dextroseagar (PDA), is transferred to a fresh plate or tube of newly prepared,sterilized medium (for example PDA) using a sterilized instrument suchas a scalpel. Any aseptic transfer of an axenic culture to fresh culturemedium achieves the objective of vegetative propagation. Thesetechniques are standard and absolutely routine in the mushroomcultivation industry.

DESCRIPTION OF THE FIGURE

FIG. 1 is a photograph showing a front view of a culture of the presentmushroom plant variety Agaricus subrufescens ‘H1X1’ growing on asubstrate of composted straw. The tallest mushroom rises about 5 inchesabove the soil layer that covers the compost.

DETAILED DESCRIPTION OF THE INVENTION

In mushroom breeding, mycelial (=vegetative) cultures of two compatibleprogenitors must come into physical contact so that one or more fusionzones can occur between the progenitors. Within those fusion zonesnuclei and organelles from the two progenitors become associated. In thebetter-studied Agaricus bisporus, a novel, hybrid mycelium ultimatelycontaining two compatible haploid nuclear types and one mitochondrialtype emerges. This novel hybrid mycelium can be isolated and propagatedto provide the new hybrid culture, which can be further subdivided andpropagated for commercial or other purposes.

In the genus Agaricus several reproductive modes exist, includingoutcrossing (=heterothallism), inbreeding/selfing (=pseudohomothallism),and haploid reproduction (=homothallism). More than one system can existwithin species or even within isolates. In the latter case, the systemis said to be amphithallic. Based on preliminary studies, Agaricussubrufescens appears to be an amphithallic species, meaning that sporesreleased by the mushroom may carry one nuclear type (=haploid spores) ortwo nuclear types (=heterokaryotic spores). Either type can participatein hybridizations.

For the present variety, single spores were isolated and germinated fromtwo examples of Agaricus subrufescens, those being (1) ‘I-101’, asingle-spore isolate obtained from material cultivated in Japan,allegedly from strain Iwade-101, which strain was reportedly developedin Japan from Brazilian germ plasm, and (2) ‘SBRFG’, a subculture of theisolate made by Kerrigan in California in 1982. These single-sporeisolates (=SSIs) were then propagated in broth and subjected to allozymeanalysis. Segregation of alleles at the PEP1 and PEP2 loci were observedin progeny of ‘I-101’, and at an Esterase locus in ‘SBRFG’. Thisdemonstrates that meiosis, recombination and partitioning ofrecombination nuclei into spores is occurring in these isolates ofAgaricus subrufescens, therefore is species is not homothallic.Furthermore, some SSIs had heteroallelic genotypes, providing thatmultiple nuclei were present in heterokaryotic spores, while otherspores were homoallelic, implying that they could be homokaryotic. Allof these observations are consistent with the presence of a basic systemof amphithalillic reproduction in which outcrossing is possible.

Homoallelic SSIs were selected from the ‘I-101’ and ‘SBRFG’ stocks to bethe progenitors of a series of hybrids. Crosses were made by placinginocula of two different SSIs, one from each parent stock, onto sterilenutrient media, and allowing the mycelia to grow into contact and fuse.Hybrid cultures were then isolated for each pairing.

The hybrid status of the putative new hybrids was verified by allozymeanalysis. In the case of the new hybrid ‘H1X1’, the progenitor‘I-101’-s1 carries allele Pep2-s, while progenitor ‘SBRFG’-s1 carriesallele Pep2-f. The hybrid ‘H1X1’ between these two SSIs has the expectedgenotype Pep2-s/f, demonstrating that this isolate incorporates DNA fromeach parent. The simplest and most conventional explanation is that‘H1X1’ received one nucleus from ‘I-101’-s1 and another nucleus from‘SBRFG’-s1.

A set of 25 Agaricus subrufescens hybrids between parents ‘I-101’ and‘SBRFG’ were grown on small containers of compost under standardconditions, with the ‘SBRFG’ parent added as a control. Hybrid ‘H1X1’was one of only two hybrids to produce a crop after only 17 days, andwas four days earlier than the ‘SBRFG’ parent. Hybrid ‘H1X1’ also hadthe highest yield, at 25% of the ‘SBRFG’ control, and 347% of theaverage of the 24 sibling hybrids. The properties of the mushroomsproduced were also desirable, as described below.

The Agaricus subrufescens hybrid ‘H1X1’ differs from its parents inseveral economically important respects. In repeated tests ‘H1X1’ hasrapidly produced an abundant crop of large mushrooms with smooth fleshyround caps. Neither the ‘I-101’-s1, the ‘SBRFG’, nor the ‘SBRFG’-s1strains produce mushrooms with this combination of traits, whereas the‘H1X1’ hybrid expresses this unique and desirable combination of traitsfrom the two parents.

A comparison of those traits among members of this pedigree is providedin Table 1.

TABLE 1 Comparison of economically important traits in members of thepedigree of the ‘H1X1’ variety Size/ Trait Strain mass Speed AbundanceCap shape ‘I-101’-s1 1x Day x 1x Flat, smooth, thin ‘H1X1’ 3x-5x Day x −7 8-10x Round, smooth, thick ‘SBRFG’ 3x-6x Day x − 3 8-9x Round,wrinkled, thick (−7) ‘SBRFG’-s1 3x-6x Day x + 5 4x Round, wrinkled,thick

The size and mass (or weight) of a typical ‘H1X1’ mushroom is on averagethree to five times greater than that of an ‘I101’-s1 mushroom, but is abit smaller than the largest mushrooms from ‘SBRFG’. The advantage oflarge mushrooms to the mushroom producer is that mushrooms of betterperceived quality and higher value are produced, and these can beharvested more rapidly and at lower cost (per unit weight picked) thancould a smaller mushroom.

Variety ‘H1X1’ typically produces a crop 7 days earlier than does‘I-101’-s1; this trait is also present in its ‘SBRFG’ parent. The yield(total weight) of a crop of ‘H1X1’ is typically 7-8 times as great asthe crop produced by ‘I-101’-s1, and may exceed that of ‘SBRFG’, aftertwo flushes of mushrooms have been harvested. Taken together, these twotraits means that the mushroom producer can produce a relatively largercrop in a shorter period of time, allowing extra crops to be plantedeach year.

The ‘H1X1’ hybrid has a very attractive cap combining the best qualitiesof the ‘Iwade 101’-s1 and ‘SBRFG parents. The round cap of ‘H1X1’ hasmore tissue mass and matures more slowly than the flat, thinner cap of‘I-101’-s1. The smooth cap surface of ‘H1X1’ is more attractive, remainscleaner and more free of soil-borne bacteria, and is easier the cleanthan wrinkled cap of ‘SBRFG’. Therefore the crop of ‘H1X1’ has morevalue per unit weight because it is cleaner and requires less cleaning,has lower losses due to bacterial discoloration associated with soiltrapped in wrinkles, and because its greater attractiveness creates aretail product that is more highly graded and priced.

A formal description of the mushrooms produced by variety ‘H1X1’follows:

Pileus at harvest stage sub-spherical to hemispherical, 50-100 mm broad,smooth, covered with tiny (ca. 1 mm×1 mm) reddish brown (about darkReddish Orange (RHS 175A-175C)) appressed scales on a whitishbackground. Flesh ca 10 mm thick. Lamellae free, close, initiallywhitish, becoming pinkish (about Pale Yellowish Pink (RHS 159C-159D)),light brown (about Yellowish Gray (RHS 156A)), and ultimately darkchocolate brown (Dark Red (RHS 187A) to Dark Grayish Reddish Brown(RHS200A) or more black) as maturation progresses. Veil forming a broad,elastic, pendant white annulus, semi-smooth above, floccose below,remaining attached to pileus margin well into maturation, leavingappendiculate remnants after dehiscence. Stipe white, smooth, subequal,20-25 mm broad above, 30-40 mm broad below, by 8-15 cm long, baseattached by mycelial cords, interior stuffed hollow; all parts becomingslightly to moderately yellow (about Light Yellow (RHS 16D) or morepale) when bruised, crushed or cut. Odor of almond extract. Chemicalreactions and microscopic features are as previously described inKerrigan, “Agaricus subrugescens, a cultivated edible and medicinalmushroom, and its synonyms,” Mycologia, 97(1), May 18, 2005, pp.12-24,and in Kerrigan, Agaricales of California Vol. 6 Agaricacae. Arcata,Calif.: Mad River Press, pp.62, and as understood by those havingordinary skill in the art for the species Agaricus subrufescens.

1. A new and distinct variety of Agaricus subrufescens mushroom assubstantially illustrated and described in the specification.